Metabolic processes have long been proposed for anabolic and catabolic bioconversions. Microorganisms of various types have been proposed for these bioconversions and include bacteria and archaea, both of which are prokaryotes; fungi; and algae. Metabolic processes are used by nature, and some have been adapted to use by man for millennia for anabolic and catabolic bioconversions ranging from culturing yogurt and fermentation of sugars to produce alcohol to treatment of water to remove contaminants. Metabolic processes offer the potential for low energy consumption, high efficiency bioconversions in relatively inexpensive processing equipment and thus may be and are often viable alternatives to chemical synthesis and degradation methods. Often anabolic processes can use raw materials that are preferred from a renewable or environmental standpoint but are not desirable for chemical synthesis, e.g., the conversion of carbon dioxide to biofuels and other bioproducts. Catabolic bioconversions can degrade substrates and have long been used for waste water treatment. Considerable interests exist in improving metabolic processes for industrial use and expanding the variety of metabolic process alternatives to chemical syntheses and degradations.
Numerous types of process techniques have been proposed for anabolic and catabolic bioconversions. These processes include the use of suspended microorganisms, i.e., planktonic processes. Also, process techniques have been disclosed where the microorganisms are located on or within a solid support.
Workers are faced with various challenges in improving metabolic processes and in providing metabolic processes that are sufficiently economically viable to be of commercial interest. Some problems may be inherent with the feedstock itself including the presence of toxins, phages, and adventitious competitive microorganisms. Other problems may arise from the microorganism to be used for the bioconversion such as low metabolic conversion rate, low population growth rate, automutation, significant consumption of substrate to support population growth, the need for inducers, co-metabolites, promoters and performance enhancing additives, and the lack of a microorganism that has the sought metabolic conversion. And yet further problems may arise from the process used for the bioconversion such as costs in recovering bioproducts from an aqueous fermentation broth. Especially with supported microorganisms, problems can arise from instability of the biofilms, including their physical degradation; overgrowth of the population of microorganisms causing suffocation; sloughing off of the microorganisms from the support; and susceptibility to competitive microorganisms. Additionally, metabolic processes are characterized as generating solid debris from dead or lysed cells, and the debris needs to be accommodated in the process to remove these solids. In some instances, the debris have value as feed supplements such as distillers grains from the manufacturing of ethanol, but in other metabolic processes such as for the treatment of municipal waste water, costs may have to be incurred to dispose of the debris in an environmentally acceptable manner. Genetic engineering, which has been proposed to overcome one or more of these problems, can itself be problematic.
Microorganisms, including but not limited to, bacteria, archaea, fungi and algae, are capable of becoming attached or adhered to a surface. Studies have been conducted pertaining to the effect of a change from planktonic growth to growth of microorganisms on surfaces, including the formation of biofilms on surfaces. A number of workers have investigated preventing or degrading biofilms in an animal or human body to enhance the efficacy of antibiotic treatments to cure the animal or human body.
Tuson, et al., in “Bacteria-surface Interactions”, Soft Matter, Vol. 1, issue 608 (2013) citable as DOI: 10.1039/c3sm27705d, provide a review of work in the field of bacterial-surface interactions. The authors describe the processes involved in attaching a microorganism to a surface and recite that attachment to surfaces causes phenotypic switches in the cells and that the surface can provide benefits to the attached cells. The authors recite that organic matter can concentrate at horizontal surfaces stimulating growth of bacteria associated with the surface, and increasing substrate surface area provides more area on which nutrients can absorb, enabling cells to grow at nutrient concentrations that would normally be too low to support growth. The authors further state that in addition to surface attachment facilitating nutrient capture, some bacteria obtain necessary metabolites and co-factors directly from the surfaces to which they adhere.
Some of the observations reported in this article include that nucleating cell growth into communities on surfaces protect cells from predation and other environmental threats and facilitate the conservation of the genotype. The authors recite that where the microorganisms form biofilms, resistance to antibiotic treatment has been observed. This resistance has been attributed to one or more of the barrier function of the biofilm matrix; the presence of dormant persister cells and highly resistant colony variants, and upregulation of several biofilm-specific antibiotic resistance genes. One group of workers have postulated that some adhering cells not associated with biofilms have resistance to antibiotics due to primary mechanisms of reducing the net negative charge on bacterial cells and enhancing the stability of the membrane. Tuson, et al., points to a conclusion drawn in one article that the attachment of bacteria to surfaces alters their metabolic state and reduces antibiotic susceptibility, which is a common feature of bacteria during the stationary phase of cell growth.
In respect of cell activities pertaining to association of bacteria with surfaces, the authors discuss that surface sensing is a precursor to swarming which is an important adaptive behavior in which contact between cells and surfaces programs morphological changes that facilitate cooperative behavior, rapid community growth, and migration of communities. The cells in bacterial communities such as swarms or biofilms interact with each other in several different ways. Bacteria are able to communicate through the use of small molecule chemical messengers in a process referred to as quorum sensing.                “The dense packing of cells in bacterial communities facilitates and increase in the concentration of small molecules that transfer information between cells and trigger physiological changes. The shape of chemical gradients in close proximity to surfaces enhances the exchange of chemical information within biofilms and communities attached to surfaces.”        
Cho, et al., in “Self-Organization in High-Density Bacterial Colonies: Efficient Crowd Control”, PLOS Biology, Vol. 5, Issue 11, November 2007, pages 2614 to 2623, relate their findings that E. coli in microchambers communicate to provide colony growth towards an escape from the confines of the microchambers without a potentially “stampede”-like blockage of the exit and to provide channels to facilitate nutrient transport into the colony.
Tuson, et al., further describe the steps for the formation of an attachment of a cell to a surface. The initial attachment is reversible and involves hydrodynamic and electrostatic interactions and the second step of the attachment is irreversible and involves van der Waals interactions between the hydrophobic region of the outer cell wall and the surface. Irreversible attachment is facilitated by the production of extracellular polymeric substance.                “Thermodynamics plays a central role in regulating the binding of bacteria to surfaces. Cells attach preferentially to hydrophilic materials (i.e., materials with a large surface energy) when the surface energy of the bacterium is larger than surface energy of the liquid in which they are suspended. The surface energy of bacteria is typically smaller than the surface energy of liquids in which the cells are suspended, and this mismatch causes cells to attach preferentially to hydrophobic materials (i.e., materials with lower surface energies). Bacteria are able to attach to a wide variety of different materials, including glass, aluminum, stainless steel, various organic polymers, and for needed materials such as Teflon™.”        
Tuson, et al., report that surface sensing triggers a variety of cellular changes. Many of the changes are morphological and facilitate attachment to surfaces. They state:                “Interestingly, the physical properties of surfaces may influence cell morphology and community structure.” . . . “Cells adhere uniformly to hydrophobic surfaces, form microcolonies, and grow into tightly packed multi-layer biofilms. Fewer cells attach to hydrophilic surfaces, and changes in cell division lead to the formation of chains of cells that are >100 μm long. These chains become loosely entangled to form relatively unstructured and less densely packed biofilms.”        
Tuson, et al., in their concluding remarks state:                “Our understanding of the interaction of bacteria was surfaces is remarkably incomplete. This topic seems ideally suited for collaborations between microbiologist and materials scientists, chemists, and engineers as it is poised to benefit from multidisciplinary approaches that are formulated to penetrate into a range of areas, including: (1) identifying the properties of surfaces that are sensed by bacteria; (2) elucidating the molecular mechanisms bacteria used to send surfaces and their biochemical responses; and (3) determining how to modulate surface properties to provoke a desired cellular response, including changes in morphology, alterations in bioenergetics, or cell death.”        
Many proposals exist for using a solid carrier or support for microorganisms to effect a plethora of anabolic and catabolic bioconversions; however, despite the potential process advantages provided by using a solid, commercial success has been limited to a relatively few applications. Proposals have been proffered for the microorganisms to be supported on the surface of a carrier or in pores of a carrier and for the microorganisms to be located within the carrier. See, for instance, Zhou, et al., “Recent Patents on Immobilized Microorganisms Technology and Its Engineering Application in Wastewater Treatment, Recent Patents on Engineering, 2008, 2, 28-35.
As a general rule, solid debris are generated as a result of the biological activity, e.g., from the instability of the biofilm formed on the carrier and from the death and deterioration of cell mass. For instance, Sato, et al., in U.S. Pat. No. 6,610,205, disclose processes for nitrifying and denitrifying organic waste water using a thermoplastic microbe carrier. The patentees assert that a single carrier can affect both bioconversions requiring aerobic and anaerobic conditions. The carrier, once formed, is contacted with activated sludge containing microorganisms. The patentees state that the nitrifying bacteria are “thickly grown” on the surface of the carrier and the denitrifying bacteria are “adsorbed onto the carrier and thereby are firmly immobilized thereon”. Their FIG. 1 depicts an apparatus using the carrier and includes settling tank 9 to remove sludge. Accordingly, such processes appear to require a means to remove debris from the support or carrier.
Several workers have formed an aqueous mixture of microorganisms and polymer as a solution, dispersion or emulsion. Some workers spray dried the mixture and others proposed crosslinking to obtain a solid structure containing microorganisms within the interior of the solid structure. The following discussion is provided as an illustration of proposals to form solid structures from an aqueous medium also containing microorganisms.
Hino, et al., in U.S. Pat. No. 4,148,689 disclose the use of microorganisms in a hydrophilic complex gel by dispersing microorganisms in a certain homogeneous sol and then gelling the mixture and treating it chemically or by drying to obtain a xerogel. The xerogel is said to possess desired strength and is composed of gelled water soluble polymer, such as natural polymers, polyvinyl alcohol, polyethylene glycol and polyethylene imine, and silica. The xerogels used in the examples appear to provide bioconversion, but at lesser activities than suspended cell fermentations. Most of the examples appear to demonstrate bioactivity over a short duration, e.g., less than 30 hours. Those examples that appear to report activity over longer durations also indicate deactivation over time. Indeed, the patentees contemplate that an advantage of their xerogel is that the polymer can be recovered and recycled upon deactivation. See column 9, lines 66 et seq.
Fukui, et al., in U.S. Pat. No. 4,195,129, disclose mixing microbial cells with photo-curable resin and irradiating the mixture to provide a cured product containing immobilized cells. The product, according to the examples, does not have the bioactivity of a free cell suspension. The patentees do not provide any data regarding the performance of the immobilized cells over a long duration.
Yamada, et al., in U.S. Pat. No. 4,546,081 disclose a process for continuous fermentation with yeast to produce alcohol. The yeast is immobilized in a thin film which is then positioned within a vessel for the fermentation. The patentees recite a number of different techniques for making the film containing the yeast. Although a process in which a mixture of yeast and polyvinyl alcohol is gelled by radiation and formed into the desired shape, no performance differences among the films prepared by the various techniques are specifically recited in the patent.
Ishimura, et al., in U.S. Pat. No. 4,727,030, have as an objective obtaining a molded, porous article containing microbial cells. They disclose a process for immobilizing enzymes or cells wherein the enzymes or cells are mixed with polyvinyl alcohol and activated carbon, and then the mixture is partially dried then molded and further dehydrated under specified conditions. The porous gel is said to have little expansion upon hydration.
In the 1990's a process was developed in Japan called the Pegasus Process, see, for instance, Stowa Pagasus/Pegazur/Bio-tube Process Sheets, Jun. 13, 2006, that uses organic gel pellets composed of a mixture of polyethylene glycol and nitrifying activated sludge. See also, U.S. Pat. No. 4,791,061 which is in the same patent family as KR9312103 referenced in this document. The pellets are said to have a diameter of 3 millimeters and a polyethylene glycol fraction of 15 percent and a microorganism fraction of 2 percent with a biofilm thickness of about 60 micrometers. The patent discloses preparing the pellets from a mixture containing an activated sludge and prepolymer and dropping the mixture into a water solution of polyvalent metal ion and persulfate to form particles with immobilized microorganisms. The process is asserted to reduce the loss in activity of the microorganisms in forming the pellets.
Chen, et al., in U.S. Pat. No. 5,290,693 immobilizing microorganisms or enzymes on beads of polyvinyl alcohol. They form a mixture of polyvinyl alcohol and microorganisms and then conduct a two stage gelation and hardening step using boric acid and then phosphoric acid or phosphate. The patentees state that their process provides strong beads without being detrimental to the microorganisms or enzymes immobilized. The examples are instructive. Example 1, for instance, pertains to making and using beads for denitrification of water containing 100 ppm potassium nitrate. They state at column 5, lines 7 to 11:                On the seventh day, denitrification rate of the immobilized microorganisms reached 0.65 mg NO31—N/g gel/h (sic), which remained unchanged until the 30th day. The biochemical vitality of microorganisms remained stable.”        
The solution used to make the beads contained about 25 g/L of denitrifying sludge microorganisms. This example appears to indicate that 7 days of growth of the population of microorganisms were required to achieve the activity, and that after 30 days, the stable activity was lost. The comparative control reported in this example, which used boric acid only to gel and harden the PVA, provided a denitrification rate of 0.55 mg NO3−—N/g gel/h and became unstable after 15 days. Examples 2 and 3 report data for continuous operations that extended for 10 and 20 days respectively. Example 4 pertains to the production of ethanol using Sacchramyces cerevisa (about 15 g/L in the mixture with the polyvinyl alcohol) and only 8 hours of use were reported with the beads containing the immobilized microorganisms being slightly inferior in ethanol production than unsupported yeast.
Nagadomi, et al., in “Treatment of Aquarium Water by Denitrifying Photosynthetic Bacteria Using Immobilized Polyvinyl Alcohol Beads”, Journal of Bioscience and Bioengineering, 87, 2, 189-193 (1999), confirm the observations of Chen, et al. They found that boric acid is deleterious to microorganisms. They also observed the growth of the population of microorganisms immobilized in alginate beads and in polyvinyl alcohol beads. The data reported by the authors did not extend much over 15 days of operation.
Willuwait, et al., in U.S. Pat. No. 7,384,777 B2, immobilize bacteria in polymeric matrices. The matrices are used for the controlled release of the microorganisms. As explained at column 3, lines 63 to 67:                “By means of the cleaning process, the microorganisms multiply until the holding capacity of the capsules/spheres or the gel has been reached and the wall bursts, i.e., the microorganisms are released.”        
It is not surprising, therefore, that the large bulk of activities directed towards improving metabolic processes have focused on changing the genotype of the microorganism, e.g., through genetic engineering. Genotypic alterations often come at significant expense and require substantial time to obtain the sought performance from a microorganism. Typically most genetically engineered microorganisms lack robustness, e.g., are slow growing and are competitively disadvantaged against invasive microorganisms and are subject to losing plasmids during scale up for quantities sufficient to fill commercial-scale bioreactors and during the bioconversion process itself. Additionally, genetically engineered microorganisms may have to be carefully contained so as not to escape to the environment, and disposal of debris from metabolic processes using genetically engineered microorganisms may be treated as hazardous waste.